3.1 An Al-tolerant Eucalyptus hybrid clone has enhanced accumulation and exudation of malate and citrate
The available evidence indicates that the Al-induced secretion of organic acids from roots may lead to the detoxification of Al in higher plants [32, 33]. A role for organic acids leading in Al tolerance in Eucalyptus has been observed previously [12, 18, 19]. The lower root tip concentration of Al coupled with the higher root secretion of citrate and malate in Al-tolerant E. grandis × E. urophylla clone G9 than that in Al-sensitive E. urophylla clone W4 suggested that secretion of these two organic acids was involved in the increased tolerance to Al in G9. This trait was consistent with the results reported for Al-tolerant E. camaldulensis [12, 18, 34]. Tahara et al. [12] documented in E. camaldulensis that citrate had the strongest capacity to bind Al among citrate, oxalate, malate and phosphate. It was likely that the Al-stimulated accumulation and secretion of citrate was the main underlying mechanism contributing to detoxification of Al in Eucalyptus roots, particularly in Al-tolerant genotypes. However, Silva et al. [19] put forward the hypothesis that Al tolerance was due to the internal detoxification of Al by complexation by malate. These conflicting conclusions left the role of malate in Eucalyptus tolerance unclear. Adding to the complexity, the types of organic acids produced and released in response to Al may vary among Eucalyptus species [34], as do the quantities, as shown here for malate and citrate. The features of malate and citrate accumulation and secretion in Al-tolerant hybrid clone G9 and Al-sensitive parental clone W4 have provided further clues for the identification Al-induced genes or proteins.
3.2 New potential carrier proteins involved in citrate secretion, but malate secretion facilitated by anion channel in E. grandis × E. urophylla
A rapid release of organic acid in response to exposure to Al would suggest that pre-existing anion transporters on the plasma membrane quickly initiated organic acid secretion without the need to produce new proteins; however, a lag in the release of organic acids could indicate that gene expression and/or protein synthesis was required [33, 35, 36]. There was no significant delay in malate secretion by G9, followed by an increase in the secretion of citrate after a lag period of more than 3 h. In contrast, in W4, there was a lag of more than an hour after Al exposure before malate secretion became apparent, while Al exposure did not induce the production or secretion of citrate. Thus, 24 h after exposure to Al, the synthesis and secretion of malate and citrate by G9 was much greater than in W4.
Both PG and CHM significantly reduced the Al-induced secretion and internal concentration of citrate in roots of two Eucalyptus clones as well as the malate concentration in G9. However, CHM had no impact on malate secretion in W4, indicating that there are different pathways operating for citrate and malate secretion in response to Al in the two clones. Generally, Al-tolerant species or genotypes had stronger induction and higher quantities of carrier proteins on membranes inside root cells and anion channel proteins on the plasma membrane of root cells, than Al-sensitive genotypes [37, 38]. If organic acid synthesis and transport need new carrier proteins involved, an obvious lag of several hours before secretion would be apparent ; an anion channel proteins already present in allow the organic acid to be secreted out of the root more quickly [39, 40]. Therefore, in G9, it seems likely that pre-existing anion channel proteins facilitated the immediate secretion of malate, while a new carrier protein apparently had to be produced before citrate could be transported out of the roots. Anion channel proteins, such as ALMT and MATE/AACT, are localized to the plasma membrane of root cells and transport their substrates to rapidly facilitate organic acid release at phytotoxic concentrations of Al3+ [41, 42]. Sawaki et al. [1] found that citrate excretion induced by Al in E. camaldulensis roots was associated with over expression of EcMATE on the plasma membrane, which led to Al tolerance. However, other components remain to be revealed, particularly the new protein-coding genes and their functions in organic acid synthesis and transport. For instance, the delay in citrate secretion found in G9 may be due to the need to produce new proteins involved in the synthesis and delivery of citric acid. For W4, there was no change in citrate secretion in response to exposure to Al, while malate secretion was delayed and did not reach its maximum level for 6 h. Moreover, CHM had no effect on the secretion of malate, but did inhibit its accumulation, indicating that W4 does not lack the capacity to release malate, but rather was restricted in its ability to produce malate.
We speculate that other organic substances might be involved in detoxifying Al in some Eucalyptus genotypes. A consequence of Al tolerance in Eucalyptus was the maintenance of nutrients and photosynthesis [17, 43]. A new low-molecular-weight Al-binding ligand from roots, oenothein b, contributed to Al tolerance in E. camaldulensis [12, 44]. In addition, some allelochemicals, such as phenolic compounds, have been found to be involved in Al detoxification by binding strongly with Al ions in the cytoplasm of woody plants including Eucalyptus viminalis Labill. [45]. Transcriptome analysis has also revealed that genes associated with flavonoid and phenylpropanoid biosynthetic pathways have key roles in the response of roots of Chinese fir to Al [46]. All these findings encourage further research to identify compounds that confer Al tolerance to hybrid clones of Eucalyptus.
3.3 The secretion and accumulation of citrate and malate in hybrid clone E. grandis × E. urophyllaGL-9 were closely linked with changes in CS and PEPC activities
We observed that CS and PEPC activities in both clones were markedly induced by Al, while ME activity significantly decreased. Together, these changes likely contribute to the increased biosynthesis of organic acids by feeding carbon skeletons into the TCA cycle [47]. The balance between synthesis or catabolism of Al-induced citrate and malate was regulated by shifts in activities of various metabolic enzymes that together contributed to accumulation of these organic acids to increase Al tolerance in Eucalyptus. Additionally, the addition of inhibitors (PG and CHM) directly or indirectly caused changes in enzyme activity involved in organic acid metabolism.
In the case of E. urophylla clone W4, decreased ME activity may play a greater role in the lower malate accumulation in the presence of Al, since MDH activity was unchanged. Meanwhile, the activities of ACO and IDH were significantly increased by Al exposure, which may result in the lack of an increase in citrate. In E. grandis × E. urophylla clone G9, increased synthesis and secretion of malate seemed to be supported by decreased ME and MDH activity to prevent malate metabolism. We speculate that genes encoding ME may contribute to increased internal malate and citrate concentrations, leading to exudation of these organic acids to confer higher Al resistance, as in soybean [26]. CS is typically regarded as the main enzyme necessary to increase synthesis and secretion of citrate in roots of Al-tolerant plants, such as rye [48], Paraserianthes facataria [49], soybean [50]. Moreover, the transcript levels for CS, ALMT and MATE in the root apex of an Al-tolerant cultivar of alfalfa were higher than in an Al-sensitive cultivar [27]. However, Ikka et al. [34] found that the Al-induced increase in citrate concentration in roots of E. camaldulensis was not due to increased CS activity, but was dependent on reducing ACO activity to suppress citrate catabolism. Recently, Teng et al. [51] reported that CS, PEPC and IDH may play important roles in organic acid biosynthesis and degradation in Eucalyptus. Our study indicated that the increased synthesis and secretion of citrate that contributed to increase Al-tolerance in E. grandis × E. urophylla was likely achieved by increasing the activities of PEPC and CS, and decreasing the activity of ACO. These three enzymes may be involved in creating the balance between the secretion of malate and citrate in the roots of plants exposed to Al.
It is clear that key enzymes regulating organic acid synthesis and exudation vary among of Eucalyptus genotypes. Some effort has been made in plants to increase the expression of enzymes such as PEPC, CS and MDH that which contribute to the balance and increase of organic acid metabolism [52-54]. The overexpression of CS and PEPC may produce a new citrate synthesis pathway [52, 55]. Alterations in the expression of the corresponding genes can affect OA synthesis and exudation resulting in changes in Al tolerance [56]. These studies indicate that the synthesis of organic acids could be increased by regulating the transcription and translation of genes encoding enzymes involved in organic acid synthesis in Eucalyptus.